Christian Schmees

Despite tremendous progress in deciphering breast cancer at the genomic level, the pronounced intra- and intertumoral heterogeneity remains a major obstacle to the advancement of novel and more effective treatment approaches. Frequent treatment failure and the development of treatment resistance highlight the need for patient-derived tumor models that reflect the individual tumors of breast cancer patients and allow a comprehensive analyses and parallel functional validation of individualized and therapeutically targetable vulnerabilities in protein signal transduction pathways. Here, we introduce the generation and application of breast cancer patient-derived 3D microtumors (BC-PDMs). Residual fresh tumor tissue specimens were collected from n = 102 patients diagnosed with breast cancer and subjected to BC-PDM isolation. BC-PDMs retained histopathological characteristics, and extracellular matrix (ECM) components together with key protein signaling pathway signatures of the corresponding primary tumor tissue. Accordingly, BC-PDMs reflect the inter- and intratumoral heterogeneity of breast cancer and its key signal transduction properties. DigiWest®-based protein expression profiling of identified treatment responder and non-responder BC-PDMs enabled the identification of potential resistance and sensitivity markers of individual drug treatments, including markers previously associated with treatment response and yet undescribed proteins. The combination of individualized drug testing with comprehensive protein profiling analyses of BC-PDMs may provide a valuable complement for personalized treatment stratification and response prediction for breast cancer.

Background Adoptive cell therapy (ACT) has shown promising results for the treatment of cancer and viral infections. Successful ACT relies on ex vivo expansion of large numbers of desired T-cells with strong cytotoxic capacity and in vivo persistence, which constitutes the greatest challenge to current ACT strategies. Here, in this study, we present a novel technology for ex vivo expansion of antigen-specific T-cells; artificial antigen-presenting scaffolds (Ag-scaffolds) consisting of a dextran-polysaccharide backbone, decorated with combinations of peptide-Major Histocompatibility Complex (pMHC), cytokines and co-stimulatory molecules, enabling coordinated stimulation of antigen-specific T-cells. Methods The capacity of Ag-scaffolds to expand antigen-specific T-cells was explored in ex vivo cultures with peripheral blood mononuclear cells from healthy donors and patients with metastatic melanoma. The resulting T-cell products were assessed for phenotypic and functional characteristics. Results We identified an optimal Ag-scaffold for expansion of T-cells for ACT, carrying pMHC and interleukin-2 (IL-2) and IL-21, with which we efficiently expanded both virus-specific and tumor-specific CD8+ T cells from peripheral blood of healthy donors and patients, respectively. The resulting T-cell products were characterized by a high frequency of antigen-specific cells with high self-renewal capacity, low exhaustion, a multifunctional cytokine profile upon antigen-challenge and superior tumor killing capacity. This demonstrates that the coordinated stimuli provided by an optimized stoichiometry of TCR engaging (pMHC) and stimulatory (cytokine) moieties is essential to obtain desired T-cell characteristics. To generate an ‘off-the-shelf’ multitargeting Ag-scaffold product of relevance to patients with metastatic melanoma, we identified the 30 most frequently recognized shared HLA-A0201-restricted melanoma epitopes in a cohort of 87 patients. By combining these in an Ag-scaffold product, we were able to expand tumor-specific T-cells from 60–70% of patients with melanoma, yielding a multitargeted T-cell product with up to 25% specific and phenotypically and functionally improved T cells. Conclusions Taken together, the Ag-scaffold represents a promising new technology for selective expansion of antigen-specific CD8+ T cells directly from blood, yielding a highly specific and functionally enhanced T-cell product for ACT.

Despite tremendous progress in deciphering breast cancer at the genomic level, the pronounced heterogeneity remains a major obstacle to the advancement of novel and more effective treatment approaches. Frequent treatment failure and the development of treatment resistance highlight the need for patient-derived tumor models that reflect the individual tumors of breast cancer patients and allow a comprehensive analyses and parallel functional validation of individualized and therapeutically targetable vulnerabilities in protein signal transduction pathways. Here, we introduce the generation and application of breast cancer patient-derived 3D microtumors (BC-PDMs). Residual fresh tumor tissue specimens were collected from n = 102 patients diagnosed with breast cancer and subjected to BC-PDMs isolation. BC-PDMs retained histopathological characteristics, and extracellular matrix (ECM) components together with key protein signaling pathway signatures of the corresponding primary tumor tissue. Accordingly, BC-PDMs reflect the intertumoral heterogeneity of breast cancer and its key signal transduction properties. DigWest®-based protein expression profiling of identified treatment responder and non-responder BC-PDMs enabled the identification of potential resistance and sensitivity markers of individual drug treatments, including markers previously associated with treatment response and yet undescribed proteins. The combination of individualized drug testing with comprehensive protein profiling analyses of BC-PDMs may provide a valuable complement for personalized treatment stratification and response prediction for breast cancer.

Background Tumor-adjacent benign mammary epithelium and myoepithelium can play a pivotal role in tumor growth and progression. We investigated the invasive behavior of patient-derived microtumors and breast cancer cell line-derived spheroids in co-culture with induced pluripotent stem cell-derived mammary-like organoids in an autologous and allogenic manner. This co-culture systems enables a better understanding of the tumor-promoting function of the benign mammary (myo-) epithelium in different types of breast cancers. Methods Using three-dimensional co-culture settings of induced pluripotent stem cell-derived mammary-like organoids and patient-derived microtumors or cancer cell line-derived spheroids, we investigated tumor growth and invasiveness of the cancers by using imaging-based analysis. Levels of Fibronectin and Metalloproteinase-2 in co-cultures and respective mono-cultures were measured using multiplexed Luminex assay. Results We observed significant increases in growth and invasiveness of invasive ductal carcinoma of no special type patient-derived microtumors in co-culture with induced pluripotent stem cell-derived mammary-like organoids. We identified upregulations of the prognostic markers Fibronectin and Metalloproteinase-2 in all co-cultures compared to respective mono-cultures of mammary-like organoids, patient-derived microtumors and cell line-derived spheroids. Conclusions These findings indicate a tumor-promoting role of the tumor-adjacent mammary (myo-) epithelium dependent on the tumor composition and tumor stage. Our results highlight the importance of breast tumor models that closely resemble the heterogenous composition of primary breast tumors.

In cancer, the complex interplay between tumor cells and the tumor microenvironment results in the modulation of signaling processes. By assessing the expression of a multitude of proteins and protein variants in cancer tissue, wide-ranging information on signaling pathway activation and the status of the immunological landscape is obtainable and may provide viable information on the treatment response. Archived breast cancer tissues from a cohort of 84 patients (no adjuvant therapy) were analyzed by high-throughput Western blotting, and the expression of 150 proteins covering central cancer pathways and immune cell markers was examined. By assessing CD8α, CD11c, CD16 and CD68 expression, immune cell infiltration was determined and revealed a strong correlation between event-free patient survival and the infiltration of immune cells. The presence of tumor-infiltrating lymphocytes was linked to the pronounced activation of the Jak/Stat signaling pathway and apoptotic processes. The elevated phosphorylation of PPARγ (pS112) in non-immune-infiltrated tumors suggests a novel immune evasion mechanism in breast cancer characterized by increased PPARγ phosphorylation. Multiplexed immune cell marker assessment and the protein profiling of tumor tissue provide functional signaling data facilitating breast cancer patient stratification.

In light of the frequent development of therapeutic resistance in cancer treatment, there is a strong need for personalized model systems representing patient tumor heterogeneity, while enabling parallel drug testing and identification of appropriate treatment responses in individual patients. Using ovarian cancer as a prime example of a heterogeneous tumor disease, we developed a 3D preclinical tumor model comprised of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) to identify individual treatment vulnerabilities and validate chemo-, immuno- and targeted therapy efficacies. Enzymatic digestion of primary ovarian cancer tissue and cultivation in defined serum-free media allowed rapid and efficient recovery of PDM, while preserving histopathological features of corresponding patient tumor tissue. Reverse-phase protein array (RPPA)-analyses of >110 total and phospho-proteins enabled the identification of patient-specific sensitivities to standard, platinum-based therapy and thereby the prediction of potential treatment-responders. Co-cultures of PDM and autologous TILs for individual efficacy testing of immune checkpoint inhibitor treatment demonstrated patient-specific enhancement of cytotoxic TIL activity by this therapeutic approach. Combining protein pathway analysis and drug efficacy testing of PDM enables drug mode-of-action analyses and therapeutic sensitivity prediction within a clinically relevant time frame after surgery. Follow-up studies in larger cohorts are currently under way to further evaluate the applicability of this platform to support clinical decision making.

Background: The metabolic enzyme nicotinamide‐N‐methyltransferase (NNMT) is highly expressed in various cancer entities, suggesting tumour‐promoting functions. We systematically investigated NNMT expression and its metabolic interactions in clear cell renal cell carcinoma (ccRCC), a prominent RCC subtype with metabolic alterations, to elucidate its role as a drug target.Methods: NNMT expression was assessed in primary ccRCC (n = 134), non‐tumour tissue and ccRCC‐derived metastases (n = 145) by microarray analysis and/or immunohistochemistry. Findings were validated in The Cancer Genome Atlas (kidney renal clear cell carcinoma [KIRC], n = 452) and by single‐cell analysis. Expression was correlated with clinicopathological data and survival. Metabolic alterations in NNMT‐depleted cells were assessed by nontargeted/targeted metabolomics and extracellular flux analysis. The NNMT inhibitor (NNMTi) alone and in combination with the inhibitor 2‐deoxy‐D‐glucose for glycolysis and BPTES (bis‐2‐(5‐phenylacetamido‐1,3,4‐thiadiazol‐2‐yl)ethyl‐sulfide) for glutamine metabolism was investigated in RCC cell lines (786‐O, A498) and in two 2D ccRCC‐derived primary cultures and three 3D ccRCC air–liquid interface models.Results: NNMT protein was overexpressed in primary ccRCC (p = 1.32 × 10^–16) and ccRCC‐derived metastases (p = 3.92 × 10^–20), irrespective of metastatic location, versus non‐tumour tissue. Single‐cell data showed predominant NNMT expression in ccRCC and not in the tumour microenvironment. High NNMT expression in primary ccRCC correlated with worse survival in independent cohorts (primary RCC—hazard ratio [HR] = 4.3, 95% confidence interval [CI]: 1.5–12.4; KIRC—HR = 3.3, 95% CI: 2.0–5.4). NNMT depletion leads to intracellular glutamine accumulation, with negative effects on mitochondrial function and cell survival, while not affecting glycolysis or glutathione metabolism. At the gene level, NNMT‐depleted cells upregulate glycolysis, oxidative phosphorylation and apoptosis pathways. NNMTi alone or in combination with 2‐deoxy‐D‐glucose and BPTES resulted in inhibition of cell viability in ccRCC cell lines and primary tumour and metastasis‐derived models. In two out of three patient‐derived ccRCC air–liquid interface models, NNMTi treatment induced cytotoxicity.Conclusions: Since efficient glutamine utilisation, which is essential for ccRCC tumours, depends on NNMT, small‐molecule NNMT inhibitors provide a novel therapeutic strategy for ccRCC and act as sensitizers for combination therapies.

Background: In cancerous tissue, a complex interplay of tumour cells with different cell types from the tumour microenvironment is causing modulations in signalling processes. By directly assessing expression of a multitude of proteins and protein variants, extensive information on signalling pathways, their activation status and the effect of the immunological landscape can be obtained providing viable information for treatment response. Methods: Protein extracted from archived breast cancer tissue from patients without adjuvant therapy was subjected to high-throughput Western blotting using the DigiWest technology. Expression of 150 proteins and protein variants covering cell cycle control, apoptosis, Jak/Stat, MAPK-, Pi3K/Akt-, Wnt-, and, autophagic signalling as well as general tumour markers was monitored in a cohort of 84 patient samples. The degree of immune cell infiltration was investigated and set against treatment outcome by integrating patient specific follow-up data. Results: Characterization of the tumour microenvironment by monitoring CD8α, CD11c, CD16 and CD68 expression revealed a strong correlation of event-free patient survival with immune cell infiltration. Furthermore, the presence of tumour infiltrating lymphocytes was linked to a pronounced activation of the Jak/Stat signalling pathway and apoptotic processes. Elevated phosphorylation of peroxisome proliferator-activated receptor gamma (PPARγ, pS112) in non-immune infiltrated tumour tissue suggests a novel immune evasion mechanism in breast cancer characterized by increased PPARγ activation. Conclusion: Multiplexed immune cell marker assessment and protein profiling of tumour tissue provides functional signalling data facilitating breast cancer patient stratification.